1. Field of the Invention
The presently disclosed and claimed inventive concept(s) relates generally to compositions useful in cancer detection and/or treatment, as well as methods of producing and using same.
2. Brief Description of the Art
Cancer of the colon is the second most frequently diagnosed malignancy in the United States, as well as the third leading cause of cancer death. Colon cancer is a highly treatable and often curable disease when localized to the bowel. Surgery is the primary treatment and results in cure in approximately 50% of patients. However, recurrence and metastases following surgery is a major problem and often is the ultimate cause of death.
Due to its proximity, cancer of the colon often metastasizes to the small intestine. The prognosis of the cancer spreading to the small intestine is related to the degree of penetration of the tumor through the bowel wall and the presence or absence of nodal involvement. These two characteristics form the basis for all staging systems developed for colon cancer. Various characteristics also assist in prognosticating colon cancer and its spread to the small intestines. For example, bowel obstruction and bowel perforation are indicators of poor prognosis. Elevated pretreatment serum levels of carcinoembryonic antigen (CEA) and of carbohydrate antigen 19-9 (CA 19-9) also have a negative prognostic significance. However, age greater than 70 years at presentation is not a contraindication to standard therapies; acceptable morbidity and mortality, as well as long-term survival, are achieved in this patient population.
Cancer cells can also originate in the small intestine. However, this is a much rarer type of cancer. Symptoms of cancer of the small intestine typically include pain or cramps in the middle of the abdomen, weight loss without dieting, a lump in the abdomen, or blood in the stool.
Cancer of the stomach, also referred to as gastric cancer, also frequently metastasizes to the small intestine due to its proximity. This cancer is often difficult to diagnose in early stages and can be in the stomach for a long time, growing to a large size before symptoms arise. In the early stages of cancer of the stomach, an individual may experience indigestion and stomach discomfort, a bloated feeling after eating, mild nausea, loss of appetite or heartburn. In more advanced stages of stomach cancer, there may be blood in the stool, vomiting, weight loss or more severe pain.
Because of the frequency of these types of cancer (approximately 160,000 new cases of colon and rectal cancer per year alone), the identification of high-risk groups, the demonstrated slow growth of primary lesions, and the better survival of early-stage lesions, screening for gastrointestinal cancers should be a part of routine care for all adults starting at age 50, especially those with first-degree relatives with colorectal cancer.
Procedures used for detecting, diagnosing, monitoring, treating and preventing cancer of the colon, small intestine and/or stomach are of critical importance to the outcome of the patient. Patients diagnosed with early stage cancer generally have a much greater five-year survival rate as compared to the survival rate for patients diagnosed with distant metastasized cancers. New diagnostic methods which are more sensitive and specific for detecting early cancer of the stomach, small intestine and colon are clearly needed.
Patients with gastrointestinal cancers are closely monitored following initial therapy and during adjuvant therapy to determine response to therapy and to detect persistent or recurrent disease of metastasis. There is clearly a need for a cancer marker which is more sensitive and specific in detecting recurrence of these types of cancer.
Stem cells are ultimately responsible for the entire cell production process in a particular tissue. They have a potential capability of large numbers of cell division and maintenance of cell replacement during the entire life of an animal (Potten et al., 2003). The epithelial cells of intestinal villi of the small intestinal mucosa are replaced within 2-3 days, and this rapid cell turnover, in addition to self-renewal by the intestinal tissue, is governed by epithelial stem cells present in the crypts of the small intestine (Okano et al., 2005). The Musashi-1 (Msi-1) gene encodes an RNA binding protein involved in early asymmetric divisions generating differentiated cells from neural stem cells or progenitor cells. Msi-1 expression was observed in the small intestine at the fourth-sixth cell position from the bottom of the crypts and in the cells in the deepest portion of the large intestine, where the possibility of stem cells is considered to be high (Okano et al., 2005; and Marshman et al., 2002).
Several lines of evidence suggest that some tumor types are maintained by a small population of self-renewing cells or “cancer stem cells”. The transformation of a normal mucosal epithelial cell to an invasive colorectal carcinoma occurs via a well-coordinated accumulation of mutations in a series of critical genes (Riehl et al., 2006). In gut, tumorigenesis arises from the stem cell population located near the base of intestine and colonic crypts (Potten et al., 2003). Msi-1 has been shown to be a positive regulator of Notch signaling through its interaction and translational repression of mammalian Numb (mNumb) messenger RNA (mRNA) (an inhibitor of Notch signaling) (Okano et al., 2002). Recently, reports have emerged showing that Msi-1 regulates neuronal development through the translational repression of p21WAF1/Cip1 (Battelli et al., 2006; Sakakibara et al., 1996; and Imai et al., 2001). Msi-1 expression in intestinal tumors of APCmin/+ mice is thought to be caused by activation of Notch signaling. However, the definitive role of Msi-1 in colon cancer and cancer progression is currently unclear.
Dysregulated expression of oncogenes and tumor suppressors is a critical regulator of tumorigenesis. Known targets that lead to a tumorigenic phenotype include cyclooxygenase (COX)-2, interleukin (IL)-8 and vascular endothelial growth factor (VEGF) (Dixon et al., 2001; Dubois et al., 1998; Wang et al., 2005). COX-2 is the rate-limiting enzyme in the production of prostaglandins (PGs), an important mediator of various cellular processes including increased proliferation, apoptosis resistance and enhanced angiogenesis (Krysan et al., 2005; Mukhopadhyay et al., 2003b). COX-2 overexpression occurs in multiple tumors, and can be observed at various stages of tumorigenesis (Eberhart et al., 1994). While transcriptional activation of COX-2 is an early event, it is also regulated at the posttranscriptional levels of mRNA stability and translation (Dixon et al., 2000).
Distinct cis-acting AU-rich elements (ARE) sequence elements located within the 3′ untranslated region (3′UTR) have been identified in the COX-2, IL-8 and VEGF mRNA that regulate mRNA stability and translation (Cok & Morrison, 2001; Dixon et al., 2001; Ristimaki et al., 1996). Specifically, the first sixty nucleotides in COX-2 3′UTR encode AREs, which regulate mRNA stability and translation (Cok & Morrison, 2001; Mukhopadhyay et al., 2003a). RNA binding protein HuR interacts with these ARE sequences to regulates the stability and translation of COX-2 mRNA (Cok & Morrison, 2001; Dixon et al., 2000). HuR is also upregulated in various cancers (Denkert et al., 2006a; Denkert et al., 2004; Erkinheimo et al., 2003; Nabors et al., 2001).
RNA binding motif protein 3 (RBM3) is a ubiquitously expressed glycine-rich protein that can bind to both RNA and DNA via an amino-terminal RNA binding domain. RBM3 was identified as a protein expressed following cold shock and was found in the complex of proteins binding to COX-2. However, the correlation of RBM3 to COX-2, IL-8 and VEGF mRNA stability, translation and cancer progression have not been demonstrated.
Defining the mechanisms that regulate stem cell fate is critical in increasing our understanding of the neoplastic process. Tumorigenesis in the gut arises specifically in the stem cell (Clarke, 2005; de Lau, 2007; and He, 2007) population located at or near the base of the intestinal and colonic crypts, while transit cell populations originating from the stem cell zone become fully differentiated and are eventually sloughed into the lumen. The short life span of transit cells, whether they are mutated or not, limits their deleterious influence in the intestinal or colonic crypt (Potten, 2003; and Booth, 2002). Because no specific gut stem cell markers have been identified definitively (Bjerknes, 2005; and Kayahara, 2003), recognizing and assaying resident intestinal stem cells is quite difficult and has raised contentious argument; however, the microcolony assay following γ-irradiation is by definition a functional evaluation of intestinal stem cell fate (Withers, 1970) and can provide a mechanism for examining the early events of tumorigenesis. Because homeostatic mechanisms of stem cell proliferation are the same processes that become dysregulated in carcinogenesis (Sancho, 2003), a complete examination of these proliferation mechanisms holds medical significance in targeting future cancer treatments; therefore, a more detailed understanding of the pathways that regulate stem cell behavior is essential.
Recently, MSI-1 (Musashi-1) has been identified as a putative stem cell marker (Potten et al., (2003) Differentiation, 71:28-41). Musashi-1 was identified as an RNA binding protein that is a translational repressor of p21. Musashi-1 regulates asymmetric division in neural precursor cells, and is expressed in intestinal crypts in the stem cell zone. Its increased expression has also been observed in tumors in APC/Min mice. However, it has not been shown to be a reliable intestinal stem cell marker.
Pancreatic adenocarcinoma has the worst prognosis of any major malignancy with a 3% 5-year survival (Hoyer et al., 2006). Major obstacles in treating pancreatic cancer include extensive local tumor invasion and early metastasis. Recently, it has been proposed that pancreatic tumors arise specifically in the stem cell population located in these tissues. There is increasing evidence that a small subset of cells termed cancer stem cells (CSCs) or cancer initiating cells (CICs) are capable of initiating and sustaining tumor growth in transplantation assays (Diehn and Clarke, 2006). CSCs share unique properties with normal adult stem cells, including the ability to self-renew and differentiate. CSCs are often refractory to current standard chemotherapeutic agents and radiation therapies, as they are designed to eradicate actively cycling cells, not slowly cycling cancer stem cells. Thus novel therapies that specifically target the cancer stem cell population, either alone or in conjunction with current strategies, may be more effective in obliterating solid tumors.
The existence of CSCs was first demonstrated in acute myelogenous leukemia (Bonnet and Dick, 1997) and subsequently verified in breast (Al-Hajj et al., 2003), pancreatic (Li et al., 2007) and brain tumors (Singh et al., 2004a; Singh et al., 2003; Singh et al., 2004b). The CD133+ subpopulations from brain tumors could initiate clonally derived neurospheres in vitro showing self-renewal, differentiation, and proliferative characteristics similar to normal brain stem cells (Singh et al., 2004a; Singh et al., 2003; Singh et al., 2004b). Furthermore, transplantation of CD133+, but not CD133−, cells into NOD/SCID mice was sufficient to induce tumor growth in vivo. In a recent study, primary human pancreatic adenocarcinomas were implanted in immunocompromised mice to assess the ability of specific cell surface markers to identify a subpopulation of pancreatic cancer cells with enhanced tumorigenic potential (Li et al., 2007). A subpopulation of CD44+CD24+ESA+ cells was identified as putative pancreatic cancer stem cells.
Tumor cell heterogeneity present in most solid tumors creates an enormous challenge for cancer eradication. Current strategies for inducing cell death generally target only the most rapidly proliferating cells within a tumor. Indeed, radiation therapy targets proliferating cells, which are the most sensitive to ionizing radiation (Cohn et al., 1997; Houchen et al., 2000; Riehl et al., 2000; Tessner et al., 1998); however, it is clear that effective tumor-eradication strategies must address the potential survival mechanisms unique to each particular cell type within the malignant population (i.e., quiescent stem cells). Currently, most traditional cancer therapies are based on their ability to kill most of the tumor population (i.e., log kill assays), but these treatments often fail to destroy cancer stem cells, which have been shown in several tumor types to be more resistant to standard chemotherapeutic agents (Li et al., 2007). This may explain why standard chemotherapy is effective in causing tumor shrinkage but often fails to prevent tumor recurrence, possibly due to the surviving cancer stem cell's ability to regenerate the tumor even after chemotherapeutic insult. This is not an unreasonable inference when one considers the gastrointestinal tract, where a single surviving intestinal stem cell is able to reconstitute an entire gastrointestinal crypt following severe genotoxic or cytotoxic injury (Bach et al., 2000).
Characterization of stem cells from the hematopoietic system, neural stem cells from the central nervous system and neural crest stem cells have emphasized the importance of specific cell surface antigens that permit the isolation of stem cells by fluorescence activated cell sorting (FACS). A candidate pancreatic stem cell, which is characterized by its expression of the neural stem-cell marker nestin and lack of established islet- and duct-cell markers, has been described in published reports (Abraham et al., 2004; Lechner et al., 2002; Zulewski et al., 2001). Furthermore, the basic helix-loop-helix transcription factor neurogenin 3 (NGN3) controls endocrine cell fate specification in uncommitted pancreatic progenitor cells. In the pancreas, NGN3-positive cells co-express neither insulin nor glucagon, demonstrating that NGN3 marks early precursors of pancreatic endocrine cells. Moreover, NGN3-deficient mice do not develop any islet cells and are diabetic. These data taken together demonstrate that NGN-3 and nestin are critical components of the pancreatic stem/progenitor cell compartment. A convincing recent study demonstrates that the adult mouse pancreas contains islet cell progenitors and that expansion of the 13 cell mass following injury induced by ligation of the pancreatic duct results in NGN3 gene expression and the ensuing differentiation of endogenous progenitor cells in a cell-autonomous, fusion-independent manner (Xu et al., 2008). These data demonstrate that functional islet progenitor cells can be induced in pancreatic ducts following injury.
Therefore, there is a need in the art for new and improved methods of detecting and preventing tumor growth, identifying and isolating cancer stem cells, and producing and using cancer stem cell models. It is to such compositions and methods that the presently disclosed and claimed inventive concept(s) is directed.